Rotavirus infection is a global problem mainly affecting children under the age of five. It results in severe gastroenteritis and in worst cases death.
Rotaviruses are members of the Reoviridae family of viruses (genus Rotavirus) that affect the gastrointestinal system and respiratory tract. The name is derived from the wheel like appearance of virions when viewed by negative contrast electron microscopy. The rotavirus is usually globular shape and is named after the outer and inner shells or double-shelled capsid structure of the same. The outer capsid is about 70 nm, and inner capsid is about 55 nm in diameter, respectively. The double-shelled capsid of the rotavirus surrounds the core including the inner protein shell and genome. The genome of the rotavirus consists of double stranded RNA segments encoding at least 11 rotavirus proteins—either structural viral proteins (VP) or nonstructural proteins (NSP; Desselberger, Virus Res 190: 75-96 (2014)).
The dsRNA codes for six structural proteins (VP) and six non-structural proteins (NSP). The structural proteins comprise VP1, VP2, VP3, VP4, VP6 and VP7. Three concentric layers are formed by the assembly of VP2, VP6 and VP7 respectively, with VP4 forming “spikes” on the surface of the virus structure. VP4 is cleaved by trypsin to VP8* and VP5*. VP8* and VP5* are proteolytic products of VP4.
VP2 is a 102 kDa protein and is the most abundant protein of the viral core. It forms the inner-most structural protein layer and provides a scaffold for the correct assembly of the components and transcription enzymes of the viral core (Lawton, 2000). VP1, the largest viral protein at 125 kDa, acts as an RNA-dependent polymerase for rotavirus, creating a core replication intermediate, and associates with VP2 at its icosahedral vertices (Varani and Allain, 2002; Vende et al., 2002). VP3, a 98 kDa protein, is also directly associated with the viral genome, acting as an mRNA capping enzyme that adds a 5′ cap structure to viral mRNAs. Together, VP1 and VP3 form a complex that is attached to the outer 5-fold vertices of the VP2 capsid layer (Angel, 2007). VP6 is a 42 kDa protein which forms the middle shell of the viral core, is the major capsid protein and accounts for more than 50% of the total protein mass of the virion (González et al., 2004; Estes, 1996). It is required for gene transcription and may have a role in encapsulation of the rotavirus RNA by anchoring VP1 to VP2 in the core, as seen in bluetongue virus, another member of the Reoviridae family. It also determines the classification of rotaviruses into five groups (A to E) with group A most commonly affecting humans (Palombo, 1999). VP6 in rotavirus group A has at least four subgroups (SG), which depend on the presence or absence of SG specific epitopes: SG I, SG II, SG (I+II) and SG non-(I+II). Groups B and C lack a common group A antigen but are also known to infect humans, while group D only affects animals e.g. chickens and cows (Thongprachum, 2010).
The two outer capsid proteins VP7, a 37 kDa glycoprotein (G) and the 87 kDa protease sensitive VP4 (P), define the virus' serotypes. These two proteins induce neutralizing antibody responses and are thus used to classify rotavirus serotypes into a dual nomenclature system, depending on the G-P antigen combination (e.g. G1 P[8] or G2 P[4]) (Sanchez-Padilla et al., 2009, Rahman et al., J Clin Microbiol 41: 2088-2095 (2003)). The VP4 protein dimerizes to form 60 spikes on the outer shell of the virus, which are directly involved in the initial stages of host cell entry. The spike protein contains a cleavage site at amino acid (aa) position 248. Upon infection, it is cleaved by the protease trypsin to produce VP5 (529 aa, 60 kDa) and VP8 (246 aa, 28 kDa) (Denisova et al., 1999). This process enhances virus infectivity (cell attachment and invasion of host cell) and stabilizes the spike structure (Glass, 2006). The VP7 glycoprotein forms the third or outside layer of the virus. At present, 27 G and 35 P genotypes are known (Greenberg and Estes, 2009). VP4 and VP7 are the major antigens involved in virus neutralization and are important targets for vaccine development (Dennehy, 2007).
The non-structural proteins (NSPs) are synthesized in infected cells and function in various parts of the replication cycle or interact with some of the host proteins to influence pathogenesis or the immune response to infection (Greenberg and Estes, 2009). The rotavirus nonstructural protein, NSP4, has been shown to have multiple functions including the release of calcium from the endoplasmic reticulum (ER; Tian et al, 1995); the disruption of the ER membranes and may play an important role in the removal of the transient envelope from budding particles during viral morphogenesis (see FIG. 1); affecting membrane trafficking from the ER to the Golgi complex with its ability to bind to micro tubules (Xu et al 2000); and function as an intracellular receptor to aid in the budding of subviral particles into the ER (Tian et al 1996).
In infected mammalian cells, rotaviruses undergo a unique mode of morphogenesis to form the complete triple-layered VP2/6/4/7 viral particles (Lopez et al., 2005). The triple-layer capsid is a very stable complex which enables faecal-oral transmission and delivery of the virus into the small intestine where it infects non-dividing differentiated enterocytes near the tips of the villi (Greenberg and Estes, 2009). Firstly, the intact virus attaches to sialic acid-independent receptors via 60 VP4 dimer spikes on the surface of the virus (Lundgren and Svensson, 2001). The 60 VP4 dimer spikes on the surface of the virus allow the virus to attach to these cell receptors. VP4 is susceptible to proteolytic cleavage by trypsin which results in a conformational change that exposes additional attachment sites on the surface of the glycoprotein for interaction with a series of co-receptors.
The multi-step attachment and entry process is, however, not clearly understood but the virus is delivered across the host's plasma membrane. The VP7 outer capsid shell which is also involved in the entry process, is removed in the process and double-layered particles (DLP) are delivered into the cell cytoplasm in vesicles (FIG. 1; prior art). The DLP escapes from the vesicle and goes into non-membrane bound cytoplasmic inclusions. Early transcription of the genome by VP1 begins in particles so that dsRNA is never exposed to the cytoplasm. RNA replication and core formation takes place in these non-membrane-bound cytoplasmic inclusions. The nascent (+) RNAs are then transported into the cytoplasm and serve as templates for viral protein synthesis. VP4 is produced in the cytosol and transported to the rough endoplasmic reticulum (RER), and VP7 is secreted into the RER. VP2 and VP6 are produced and assemble in the cytosol in virosomes and subsequently bud into the RER compartments, receiving a transient membrane envelope in the process (Lopez et al., 2005; Tian et al., 1996). In the RER, the transient envelopes of the viral particles are removed and replaced by VP4 and VP7 protein monomers, with critical involvement of rotaviral glycoprotein NSP4 (Tian et al., 1996; Lopez et al., 2005; Gonzalez et al., 2000). NSP4 functions as an intracellular receptor in the ER membrane and binds newly made subviral particles and probably also the spike protein VP4 (Tian et al., 1996). NSP4 is also toxic to humans and is the causative agent of the diarrhea. The complete, mature particles are subsequently transferred from the RER through the Golgi apparatus to the plasma membrane for secretion (Lopez et al., 2005).
A variety of different approaches have been taken to generate a rotavirus vaccine suitable to protect human populations from the various serotypes of rotavirus. These approaches include various Jennerian approaches, use of live attenuated viruses, use of virus-like particles, nucleic acid vaccines and viral sub-units as immunogens. At present there are two oral vaccines available on the market, however, these have low efficacy in due to strain variation.
U.S. Pat. Nos. 4,624,850, 4,636,385, 4,704,275, 4,751,080, 4,927,628, 5,474,773, and 5,695,767, each describe a variety of rotavirus vaccines and/or methods of preparing these vaccines, where the whole viral particles is used to create each of the rotavirus vaccines.
Production of rotavirus-like particles is a challenging task, as both the synthesis and assembly of one or more recombinant proteins are required. Rotavirus comprises a capsid formed by 1860 monomers of four different proteins. For RLP production the simultaneous expression and assembly of two to three recombinant proteins may be required. For example, an inner layer comprising 120 molecules of VP2, 780 molecules of VP6 (middle layer) and an outer layer of 780 molecules of the glycoprotein VP7 and 60 VP4 dimers, to form a double or triple-layered particle (Libersou et al. J. of Virology, March 2008).
Crawford et al. (J Virol. 1994 September; 68(9): 5945-5952) describe the expression of VP2, VP4, VP6, and VP7 in a baculovirus expression system. Co-expression of different combinations of the rotavirus major structural proteins resulted in the formation of stable virus-like particles (VLPs). The co-expression of VP2 and VP6 alone or with VP4 resulted in the production of VP2/6 or VP2/4/6 VLPs, which were similar to double-layered rotavirus particles. Co-expression of VP2, VP6, and VP7, with or without VP4, produced triple-layered VP2/6/7 or VP2/4/6/7 VLPs, which were similar to native infectious rotavirus particles. The VLPs maintained the structural and functional characteristics of native particles, as determined by electron microscopic examination of the particles, the presence of non-neutralizing and neutralizing epitopes on VP4 and VP7, and hemagglutination activity of the VP2/4/6/7 VLPs.
Vaccine candidates generated from rotavirus-like particles of different protein compositions have shown potential as subunit vaccines. O'Neal et al. (J. Virology, 1997, 71(11):8707-8717) show that VLPs containing VP 2 and VP6, or VP2, VP6, and VP7, and administered to mice with and without the addition of cholera toxin induced protective immunity in immunized mice. Core-like particles (CLP) and VLPs have also been used to immunize cows with VLPs more effective than CLPs in inducing passive immunity Fernandez, et al., (Vaccine, 1998, 16(5):507-516).
Plants are increasingly being used for large-scale production of recombinant proteins. For example US 2003/0175303 discloses the expression of recombinant rotavirus structural protein VP6, VP2, VP4 or VP7 in stably transformed tomato plants.
Saldana et al. (Viral Immunol. 19: 42-53 (2006)) expressed VP2 and VP6 in the cytoplasm of tomato plants. Electron microscopy studies showed that a small proportion of the proteins had assembled into 2/6 VLPs. A protective immune response was detected in mice and this may have to some extent been contributed by the non-assembled VPs. Individual proteins have been shown to elicit immune responses in mice, as in the case of VP8 and VP6 (Rodriguez-Diaz et al. Biotechnol Lett. 2011, 33(6):1169-75, Zhou et al., Vaccine 28: 6021-6027 (2010)).
Matsumura et al., (Archives of Virology 147: 1263-1270 (2002)) report bovine rotavirus A VP6 expression in transgenic potato plants. The VP6 was expressed, purified and immunogenic studies performed Immune-response in adult mice showed presence of VP6 antibodies in the sera. However, no evidence of assembled VP6 proteins was provided. It may have been that monomers or trimers of VP6 were responsible for eliciting the immune response. O'Brien et al. (2000, Virol. 270: 10444-10453) show VP6 assembly in Nicotiana benthamiana using a potato virus X (PVX) vector. Assembly of VP6 protein into icosahedral VLPs was only observed when the VP6 was fused to the PVX protein rods. Following cleavage the VP6 assembled into the icosahedral VLPs.
Codon-optimized human rotavirus VP6 has been successfully expressed in Chenopodium amaranticolor using a Beet black scorch virus (BBSV) mediated expression system. The protein was engineered as a replacement to the coat protein of BBSV. Oral immunization of female BALB/c mice with the plant based VP6 protein induced high titers of anti-VP6 mucosal IgA and serum IgG (Zhou et al., Vaccine 28: 6021-6027 (2010)). However, there was no teaching that the VP6 proteins assembled into VLPs or particles.
Rotavirus VP7 has been expressed in potato plants and was shown to produce a neutralizing immune response in mice (Yu and Langridge, 2001 Nature Biotechnol 19: 548-552). In transgenic potato plants, the VP7 gene was stable over 50 generations, with the VP7 protein from the 50th generation induced both protective and neutralizing antibodies in adult mice (Li et al., 2006, Virol 356:171-178).
Yang et al. (Yang Y M, Li X, Yang H, et al. Science China Life Science 54: 82-89 (2011)) co-expressed three rotavirus capsid proteins VP2, VP6 and VP7 of group A RV (P[8]G1) in tobacco plants and expression levels of these proteins, as well as formation of rotavirus-like particles and immunogenicity were studied. VLPs were purified from transgenic tobacco plants and analyzed by electron microscopy and Western blot. These results indicate that the plant derived VP2, VP6 and VP7 protein self-assembled into 2/6 or 2/6/7 rotavirus like particle with a diameter of 60-80 nm.
WO 2013/166609 described the production of rotavirus-like particle (RLPs) in plants, by co-expressing rotavirus structural proteins VP2, VP4, VP6 and VP7 in plants and purifying the resulting RLPs in the presence of calcium.
Rotavirus NSP4 has been expressed and purified from insect cells (Tian et al. 1996, Arch Virol. 1996; Rodriguez-Diaz et al. Protein Expr. Purif. 2003) and in E. coli (Sharif et al. Medical Journal of the Islamic Republic of Iran 2003). NSP4 has also been expressed as a fusion protein with the cholera toxin B (CTB) subunit in potato (Arakawa et al., Plant Cell Report 20: 343-348 (2001)).